Small molecule compounds targeting pgk1 and uses thereof

Abstract

The present application belongs to the technical field of tumor targeted therapy, and particularly relates to an anti-hepatoma small molecule compound targeting PGK1 and application thereof. The present application provides an anti-hepatoma small molecule compound targeting PGK1 or a pharmaceutically acceptable salt thereof. The present application provides a spirocyclic alkaloid compound with anti-tumor activity, which can be applied to the development of a PGK1 targeting inhibitor for resisting hepatoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tumor targeted therapy, and particularly relates to an anti-hepatocarcinoma small molecule compound targeting PGK1 and application thereof. BACKGROUND

[0002] Phosphoglycerate kinase 1 (PGK1) is the first ATP generating enzyme in the glycolysis process, and the reaction process catalyzed thereby is an important part of cell metabolism, and provides necessary energy and material basis for cell growth and proliferation. Overexpression and high activity of PGK1 play an important role in the occurrence, development and poor prognosis of tumors, and PGK1 is an important target in the field of tumor metabolic drug research, but there is no PGK1 inhibiting drug on the market at present.

[0003] Hepatocarcinoma is one of the common malignant tumors in the world, and the morbidity and mortality of hepatocarcinoma rank in the front in all cancers. Due to the characteristics such as easy recurrence, poor prognosis and drug resistance of hepatocarcinoma, the clinical treatment effect is not ideal. From the mechanism of action, hepatocarcinoma systemic therapy can be divided into two categories, one is immunotherapy, and the other is molecular targeted therapy. Molecular targeted therapy refers to the use of specific monoclonal antibodies or kinase inhibitors to target the key molecules in the hepatocarcinoma signal pathway, and to specifically block the signal transduction of hepatocarcinoma, so as to inhibit the growth and proliferation of cancer cells, and to play an anticancer effect. Among them, sorafenib as a representative of molecular targeted drugs has become a first-line drug for hepatocarcinoma treatment. However, the side effects of drugs and tumor drug resistance limit many patients to benefit from the above-mentioned therapies, and the discovery of new therapeutic targets and the research of corresponding targeted drugs are imminent. SUMMARY

[0004] The present application provides a kind of spirodine compound.

[0005] The present application provides the application of the above-mentioned compound.

[0006] According to the spirodine compound of the embodiment of the present application, the compound is selected from

[0007]

[0008] Or a pharmaceutically acceptable salt thereof.

[0009] Compound II is the acetylated precursor of compound I, which is metabolized in the plasma in vivo and hydrolyzed into the active form of compound I.

[0010] The present application provides the application of the above-mentioned compound in preparing a drug for treating cancer, preferably, the cancer is hepatocarcinoma.

[0011] The present application also provides a pharmaceutical composition comprising the above-mentioned compound and a pharmaceutically acceptable carrier.

[0012] The present application also provides a pharmaceutical composition for treating cancer, comprising the above-mentioned compound and a pharmaceutically acceptable carrier.

[0013] The spiro alkaloid compound of the present application inhibits the growth of cancer cells by targeting the metabolism-related kinase PGK1. In addition to being related to various tumors, the metabolism-related kinase PGK1 is also related to vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, amyotrophic lateral sclerosis, inflammation, and viral infection. Therefore, the spiro alkaloid compound of the present application can be used to prepare a drug for treating diseases related to vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, amyotrophic lateral sclerosis, inflammation, and viral infection.

[0014] Preferably, the present application also provides the use of the above-mentioned compound in the preparation of a PGK1-targeted inhibitory drug for treating diseases related to the metabolism-related kinase PGK1, such as vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, amyotrophic lateral sclerosis, inflammation, and viral infection.

[0015] According to an embodiment of the present application, a PGK1-targeted inhibitory drug comprises the above-mentioned compound and a pharmaceutically acceptable carrier.

[0016] Therefore, the present application also provides a method for analyzing the PGK1-targeting of the compound of formula I.

[0017] The above-mentioned drug can be prepared into an oral preparation or an injection; the oral preparation includes, but is not limited to, capsules, tablets, granules, and oral liquids; the injection includes, but is not limited to, sterile powder for injection, aqueous injection, and sodium chloride or glucose intravenous infusion.

[0018] In the above-mentioned drug, the oral preparation comprises an additive selected from at least one of a filler, a diluent, a disintegrant, a binder, a lubricant, a glidant, a surfactant, a solvent, a flavoring agent, a stabilizer, a coloring agent, and a preservative.

[0019] The filler or diluent includes sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, and alpha-starch; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium silicate aluminate.

[0020] The lubricant or glidant or anti-adherent includes stearic acid; metal stearates such as calcium stearate or magnesium stearate; talc; colloidal silicon dioxide; micro-powder silica gel; hydrogenated vegetable oil; polyethylene glycol; lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; and silicates such as silicic anhydride or silicate hydrate.

[0021] The binder includes distilled water, different concentrations of ethanol, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, and compounds similar to the above-mentioned excipients.

[0022] The disintegrant includes cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or cross-linked sodium carboxymethyl cellulose; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0023] The surfactant includes Tween-80, sodium dodecyl sulfate, sodium stearate sulfonate, and the like.

[0024] The antioxidant includes sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid, and the like.

[0025] The preservative or antibacterial agent includes benzoic acid and sodium benzoate, sorbic acid, ethanol, p-hydroxybenzoic acid esters (nipagin), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cassia oil, and peppermint oil, and the like.

[0026] The flavoring agent includes sweeteners such as sodium saccharin, aspartame, sugar syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, and the like; sour flavoring agents such as citric acid, malic acid, or tartaric acid; and aromatic agents such as fennel oil, peppermint oil, menthol, peppermint water, cassia oil, lemon essence, lemon oil, and various flavors of spices, and the like.

[0027] The method of using the above-mentioned drug includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection, or transdermal penetration, and is applied to a patient in need of treatment.

[0028] A pharmaceutically effective amount means an amount sufficient to treat a disease with an acceptable benefit / risk ratio obtainable by applying a drug. The level of effective dose can be determined depending on some factors, including the type of disease, the severity of the patient, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the discharge rate, the treatment cycle, the simultaneous use of drugs, and other factors known in the medical field. The drug of the present application can be administered as an independent therapeutic agent, or in combination with other therapeutic agents. Also, the composition of the present application can be added continuously or simultaneously with typical therapeutic agents, and the composition can be administered once or multiple times. It is important to administer the minimum dose that can produce the maximum effect without side effects, taking into account all the above factors, which can be determined by a physician depending on the patient's condition, age, etc.

[0029] The present application also provides a method for preparing the above-mentioned compound:

[0030]

[0031] The method for preparing Compound I according to the specific embodiment of the present application comprises the following steps:

[0032] (1) Asymmetric Michael addition occurs in an organic solvent with 2-methoxy-p-benzoquinone A and 3-substituted isatin derivative B as reactants and chiral β-ICD as a catalyst to obtain intermediate C.

[0033] (2) Acetalization occurs between intermediate C and methanol under acidic conditions with methanol as a solvent to obtain Compound I.

[0034] The method for preparing Compound II according to the specific embodiment of the present application comprises the following steps:

[0035] (1) Asymmetric Michael addition occurs in an organic solvent with 2-methoxy-p-benzoquinone A and 3-substituted isatin derivative B as reactants and chiral β-ICD as a catalyst to obtain intermediate C.

[0036] (2) Acetalization occurs between intermediate C and methanol under acidic conditions with methanol as a solvent to obtain Compound I.

[0037] (3) Compound I reacts with acetic anhydride with dichloromethane as a solvent and triethylamine as an acid-binding agent to obtain the target Compound II.

[0038] The present application has the following beneficial effects:

[0039] Compound I of the present application has a significant inhibitory effect on liver cancer cells in vitro.

[0040] Compound I of the present application can directly bind to PGK1 and inhibit its kinase activity.

[0041] The compound I of the present application plays an anticancer role by inhibiting the activity of PGK1.

[0042] The spiro alkaloid compound II of the present application can be hydrolyzed into its active form I after incubation with plasma, providing a feasible solution for drug development which takes into account the in vitro stability and in vivo activity. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0044] Figure 1 The compound I shows in vitro inhibition of the proliferation of various liver cancer cells. (Left) Proliferation curve of liver cancer cells after the action of compound I (take HepG2 cells as an example, 72h); (Right) IC of compound I acting on various liver cancer cells and normal liver cells for different times 50 .

[0045] Figure 2 The compound I shows inhibition of the kinase activity of PGK1. (Left) Change curve of NADH consumption within 30min after the action of different concentrations of compound I with time; (Right) Inhibition rate of different concentrations of compound I on PGK1 calculated by OD value at reaction termination (30min).

[0046] Figure 3 The compound I shows CETSA binding analysis with PGK1 protein molecules of cells. (Left) Western-blot analysis of PGK1 protein expression; (Right) Gray scale analysis of relative expression amount of PGK1 protein, taking gray value of GAPDH protein as internal control, and normalizing gray values of two groups at 37.4℃.

[0047] Figure 4 The compound I shows SPR binding analysis with PGK1 protein molecules.

[0048] Figure 5 The compound I shows liver cancer inhibition activity dependent on the expression of PGK1. (Left) Western-blot analysis of PGK1 knockout efficiency of HepG2 and Huh7 cells; (Right) SRB method analysis of cytotoxicity of compound I acting on HepG2, Huh7, HepG2-sgPGK1, Huh7-sg PGK1 cells for 72h.

[0049] Figure 6The compound II shows the liver cancer treatment effect in vivo. (A) tumor tissue picture; (B) tumor growth curve; (C) tumor weight; (D) body weight curve. *p<0.05, **p<0.01, ns no significant difference compared with the control group.

[0050] Figure 7 The compound II can be hydrolyzed into the compound I after being incubated with plasma. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0052] The experimental materials involved in the embodiments of the present application are as follows:

[0053] Fetal bovine serum was purchased from Yixing Biotechnology Co., Ltd.

[0054] MEM medium was purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0055] DMEM medium was purchased from Jinuo Biomedicine Technology Co., Ltd.

[0056] HepG2 and Huh7 cells were purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0057] Calcium phosphate virus packaging kit (Biyun Tian), BCA protein quantification kit (Bi Yun Tian), ECL chemiluminescence detection kit, protein-free rapid blocking solution (Yaenzyme), HRP-labeled secondary antibody (Yaenzyme) were purchased from Qingdao Aifute Biological Technology Co., Ltd.

[0058] Balb / c nude mice were purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd.

[0059] His-PGK1-pEGFP recombinant plasmid, LentiCRISPRv2 sg PGK1 recombinant plasmid, psPAX2 plasmid and pMD2.G plasmid were constructed and preserved in the laboratory.

[0060] Human PGK1 monoclonal antibody, Nickel-Beads nickel magnetic agarose bead protein purification kit (Beaver), PMSF, 3-phosphoglyceraldehyde, ATP, NADH and puromycin were purchased from Shanghai Youningwei Biological Technology Co., Ltd.

[0061] Example 1 Synthesis of Spiroalkaloid Compound I

[0062]

[0063] To the reaction bottle, 3-substituted isatin derivative B (115.7 mg, 0.3 mmol), β-ICD (1 mg, 0.003 mmol), dichloromethane (2 mL) were added successively, stirred for 10 minutes at -40 °C, then 2-methoxy-p-benzoquinone A (41.4 mg, 0.3 mmol) was added, continued to react for 5 hours at -40 °C, detected by TCL, the raw material B disappeared, transferred to room temperature, removed dichloromethane by concentration under reduced pressure, obtained the crude product C.

[0064] C was redissolved in methanol (2 mL), p-toluenesulfonic acid (5.1 mg, 0.03 mmol) was added, stirred for 3 hours at room temperature, detected by TCL, the intermediate C disappeared, after concentration under reduced pressure, white solid compound I (96.7 mg, yield 60%) was obtained by silica gel column chromatography (eluent petroleum ether: ethyl acetate = 6:1).

[0065] 1 H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.64 (s, 1H), 7.53-7.47 (m, 1H), 7.38-7.28 (m, 3H), 7.16 (t, J = 7.6 Hz, 1H), 6.64 (s, 1H), 6.13 (s, 1H), 5.21 (s, 1H), 3.88 (s, 3H), 3.15 (s, 3H), 2.70 (d, J = 14.3 Hz, 1H), 2.43 (d, J = 14.3 Hz, 1H), 1.61 (s, 9H) ppm.

[0066] 13 C NMR (125 MHz, CDCl3) δ 178.19, 149.32, 147.28, 144.67, 142.44, 140.99, 139.22, 134.69, 134.63, 129.93, 128.80, 128.59, 127.06, 126.85, 125.14, 124.65, 115.27, 114.83, 112.55, 101.56, 99.55, 84.65, 56.20, 50.33, 50.22, 45.11, 28.19 ppm.

[0067] HRMS: [M+Na] + calcd. For C 29 H 28 35 ClNNaO7+ 560.1447, found: 560.1445; [M+Na] + calcd. for C 29 H 28 37 ClNNaO7 + 562.1417; found: 562.1418. [α] D 20 +16.28 (c = 0.86 in CHCI3).

[0068] HPLC analysis: DAICEL CHIRALPAK IA, n-hexane / isopropanol = 90 / 10, 1.0 mL / min, λ = 240 nm, t major = 6.45 min, t minor = 9.77 min, ee = 98%.

[0069] Example 2 Synthesis of Spiro-biological alkaloid II

[0070]

[0071] Compound I (96.7 mg, 0.18 mmol) was dissolved in dichloromethane (2 mL), triethylamine (37 μL, 0.27 mmol), acetic anhydride (21 μL, 0.22 mmol) and DMAP (2 mg, 0.018 mmol) were added at 0 °C with stirring, stirring at 0 °C for 1 hour until compound I disappeared. Water (2 mL) was added to quench the reaction, extracted with dichloromethane (3 X 3 mL), combined organic layers, dried over sodium sulfate, concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (eluent petroleum ether: ethyl acetate = 5: 1) to obtain white solid compound II (88.6 mg, yield 85%).

[0072] 1 H NMR (400 MHz, CDC13) δ 7.85 (dd, J = 8.2, 1.1 Hz, 1H), 7.59 (dd, J = 7.6, 1.4 Hz, 1H), 7.56 (q, J = 1.2 Hz, 1H), 7.44 - 7.39 (m, 1H), 7.30 - 7.23 (m, 3H), 7.09 (td, J = 7.6, 1.1 Hz, 1H), 6.65 (s, 1H), 6.17 (s, 1H), 3.76 (s, 3H), 3.10 (s, 3H), 2.62 (d, J = 14.3 Hz, 1H), 2.38 (d, J = 14.4 Hz, 1H), 2.12 (s, 3H), 1.54 (s, 9H) ppm.

[0073] 13C NMR (100 MHz, CDC13) δ 177.8, 168.6, 151.8, 149.8, 149.2, 142.0, 139.0, 135.0, 134.6, 134.1, 129.9, 128.8, 128.6, 127.2, 126.7, 125.1, 124.5, 121.3, 114.7, 114.6, 102.7, 99.7, 84.7, 56.1, 50.5, 50.0, 45.1, 28.0, 20.6 ppm.

[0074] HRMS: [M+H] + calcd for C 31 H 31 35 ClNO8 + 580.1733, found: 580.1730; [M+H] + calcd for C 31 H 31 37 ClNO8 + 582.1704, found: 582.1707. [a] D 20 +24.35 (c = 2.30 in CHCI3).

[0075] HPLC analysis: DAICEL CHIRALPAK AD-H, n-hexane / isopropanol = 90 / 10, 1.0 mL / min, λ = 210 nm, t major = 4.16 min, t minor = 4.91 min, ee = 94%.

[0076] Example 3

[0077] 1. SRB method to detect the proliferation rate of liver cancer cells

[0078] (1) trypsin digestion of liver cancer cells, resuspended in medium to single cell suspension.

[0079] (2) after counting, seed 96-well plates, single hole volume is 90 μL, cell density is 4 x 10 3 / well, placed in 37 °C, 5% CO2 incubator, cultured to complete adhesion.

[0080] (3) Different concentrations of 1557-Ac were added to the compound groups (final concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4, 0.2, 0.1 and 0.05 μmol / L, respectively), while the blank control group was added with DMSO diluted proportionally, 10 μL / well, for 24, 48, 72 and 96 h.

[0081] (4) Detection of OD515 value by SRB method:

[0082] a. Discard the culture medium in the 96-well plate, add 10% pre-cooled TCA (100 μL per well) to fix the cells, and place in a 4°C freezer for at least 1 hour.

[0083] b. Wash with ddH2O more than 5 times and let it air dry at room temperature.

[0084] c. Add 100 μL of 4 mg / mL SRB solution to each well, stain for 15 min at room temperature in the dark, and then discard the SRB.

[0085] d. Wash with 1% glacial acetic acid more than 5 times, and let it air dry at room temperature.

[0086] e. Add 150 μL of 10 mmol / L Tris-base solution to each well until the stain is completely dissolved.

[0087] (5) Place the well plate at 515nm on the microplate reader to measure the OD value.

[0088] Calculate cell proliferation rate using the following formula:

[0089] Cell proliferation inhibition rate (%) = (OD) 对照组 -OD 给药组 ) / OD 对照组 ×100%

[0090] (6) Plot the graph using GraphPad and calculate the half-maximal inhibitory concentration (IC50). 50 .

[0091] The results are as follows Figure 1 As shown, this result indicates that compound I inhibits the proliferation of various liver cancer cells in vitro, with the effect being time- and concentration-dependent. Specifically, it has a significant effect on the IC50 of HepG2 cells. 50 The concentration was 2.24 μM, and the inhibitory rate against normal L-02 hepatocytes was weak, with an IC50 > 20 μM. This indicates that compound I has a certain degree of safety in its anticancer effect.

[0092] 2. PGK1-catalyzed reaction kinetic model analysis of the PGK1 inhibitory effect of the compound.

[0093] (1) The reaction system contained: 5 mM glyceraldehyde-3-phosphate; 1 mM ATP; 5 U GAPDH protein; 0.1 mM NADH and 5 ng purified PGK1 protein, with a total volume of 100 μL. The reaction buffer was 50 mM Tris-HCl (pH 7.5) containing 1 mM MgCl2.

[0094] (2) Add all reactants except PGK1 protein to a 96-well clear culture plate, replenish the remaining volume with reaction buffer, including compound I at gradient concentrations (diluted with reaction buffer), and finally add purified PGK1 protein to mark the start of the catalytic reaction.

[0095] Place the well plate in a microplate reader at 340 nm and observe continuously for 30 min. Analyze the PGK1 catalytic activity using the OD value change curve and calculate the compound inhibition rate.

[0096] Inhibition rate (%) = (OD) 对照组 -OD 给药组 ) / OD 对照组 ×100%

[0097] (3) Plot the graph using Graphpad and calculate the half-maximal inhibitory concentration (IC50). 50 .

[0098] The results are as follows Figure 2 As shown, this result indicates that compound I directly inhibits the catalytic activity of PGK1 at the molecular level, IC50. 50 The value is 3.2 μM.

[0099] 3. Cellular thermal shift analysis (CETSA) of the interaction between the compound and PGK1 protein

[0100] (1) HepG2 cells (5×10 5 Each well was seeded into a 6-well plate and allowed to adhere for 24 hours. After 24 hours, compound I (20 μM) was added and incubated for 3 hours.

[0101] (2) Resuspend the cells treated with the compound in PBS and divide them into several equal parts. Add DMSO to 4 equal parts and add 20 μM compound I to the other 4 equal parts.

[0102] (3) Use the gradient heating program of the PCR instrument to heat the cell samples at different temperatures.

[0103] (4) After heating, the cells were frozen and thawed three times with liquid nitrogen for 3 minutes each time, and centrifuged at 20000g at 4℃ for 20 minutes.

[0104] (5) Collect the supernatant, add protein loading buffer, and boil for 15 min. Western blotting was used to detect PGK1 protein levels.

[0105] The interaction of the compound with the protein can help PGK1 to have stronger stability in a high-temperature environment. By comparing the degradation of PGK1 protein with temperature rising in the DMSO group and the compound treatment group, it can be judged whether the compound I interacts with PGK1.

[0106] The results are shown in Figure 3 As can be seen from the figure, the addition of compound I delays the degradation of PGK1 caused by temperature rise, indicating that compound I can bind to the PGK1 protein of the cell.

[0107] 4. Surface plasmon resonance (SPR) analysis of the interaction of the compound with PGK1 protein

[0108] (1) The Biacore X-100plus instrument (GE) was used to determine SPR, and the PGK1 protein was immobilized on the sensor chip (CM5) according to the standard protocol using amine coupling method.

[0109] (2) Compound I was diluted with PBS P. The binding experiment was carried out at 25°C with a flow rate of 30 μL / min using PBS P buffer as the mobile phase. The 1:1 Langmuir binding model was used, and the binding affinity constant was obtained by BIA evaluation software.

[0110] The results are shown in Figure 4 This result confirms that compound I can bind to PGK1 protein at the molecular level, and the binding strength (KD value) is 3.7 μM.

[0111] 5. Analysis of PGK1-mediated anti-hepatocarcinoma activity of the compound

[0112] (1) PGK1 was knocked out by CRISPR / Cas9 technology to construct a hepatocarcinoma stable cell line, and a three-plasmid (LentiCRISPRv2 sg PGK1, psPAX2 and pMD2.G) system lentivirus vector system was used for packaging and concentrating PGK1 knockout virus.

[0113] (2) PGK1 knockout virus was packaged by calcium phosphate transfection method according to the instruction method, and the plasmid ratio of LentiCRISPRv2 sg PGK1, psPAX2, pMD2.G was 4:3:1.

[0114] (3) Virus infection and screening of stable strains

[0115] HepG2 and Huh7 cells were inoculated in 24-well plates, 15 μl of virus packaged in the above experiment was added, 2 μL of Polybrene (4 mg / ml) was added, and then mixed and placed in the cell culture box for infection. After 12 h, the cells were changed, and the culture was continued. After 48 h of culture, the infected cells were screened for stable strains. Since the virus vector carries puromycin resistance, puromycin 4 μg / mL was continuously added to the cell culture system for continuous screening. After two weeks, no cell death was observed, and the stable cell strain with successful PGK1 knockout was obtained. The screened stable strain was used for Western Blot experiment to detect the expression level of PGK1 protein to determine the knockout efficiency, and then used for subsequent experiments.

[0116] (4) SRB method was used to analyze the cytotoxic effect of compound I on PGK1 knockout stable strains (Hun7-sgPGK1, HepG2-sgPGK1) and corresponding parent cells.

[0117] The results are shown in Table 1. Figure 5 As shown in Table 1, after PGK1 knockout, the tumor inhibitory effect of compound I was significantly weakened, and the 72 h cytotoxicity of compound I on HepG2 cells increased from 2.24 μM to 20.39 μM, and the 72 h cytotoxicity of compound I on Huh7 cells increased from 1.16 μM to 24.93 μM. The proliferation inhibitory effect of compound I disappeared, indicating that PGK1 played a key role in the anti-cancer activity of compound I. Based on the above research, it can be concluded that compound I exerts anti-hepatoma function by directly targeting PGK1.

[0118] 6 Analysis of the in vivo liver cancer treatment effect of compound I

[0119] (1) Huh7 cell xenograft tumor model in nude mice

[0120] Huh7 cells were subcutaneously inoculated in 6-week-old Balb / c-Nude mice (3 x 10 6 per mouse), and the tumor growth was monitored. When the tumor volume reached 100 mm 3 , treatment was started.

[0121] (2) The treatment group was compound I (low, medium and high doses, once a day), and the control group was normal saline (solvent), and the drug was administered for 15 days.

[0122] (3) Analysis of treatment effect:

[0123] a. Monitor the change of tumor volume and body weight, and calculate the volume according to the following formula:

[0124] Tumor volume = long diameter x short diameter 2 x 1 / 2;

[0125] b. At the termination time, the mice were sacrificed and dissected to obtain the tumor volume and weigh it;

[0126] c. Tumor volume and tumor weight directly reflect the in vivo therapeutic effect of compound I. The tumor inhibition rate is calculated using the following formula:

[0127] Inhibition rate (%) = (tumor volume) 对照组 -tumor volume 治疗组 ) / tumor volume 对照组 ×100%

[0128] Changes in animal weight and organ indices can indicate whether there are obvious toxic side effects, thus demonstrating its safety.

[0129] The results are as follows Figure 6 As shown, compound I significantly inhibited the growth of Huh7 xenograft tumors at medium and high doses, resulting in a significant reduction in tumor volume and weight. The tumor inhibition rate was 52% at 50 mg / kg and 79% at 100 mg / kg. Furthermore, the body weight of the mice in the treatment group was not different from that in the control group, indicating that the treatment at this dose was relatively safe.

[0130] 7. LC-MS study on the hydrolysis of compound II in plasma

[0131] (1) Preparation of reference stock solution

[0132] Accurately weigh compound I and compound II, dissolve them in DMSO, and prepare a stock solution with an initial concentration of 10 mmol / L. Store at -20℃.

[0133] (2) Sample pretreatment

[0134] a. Plasma collection: Take one SPF-grade nude mouse, collect blood from its heart and place it in an anticoagulant tube. Centrifuge at 3000×g for 10 min at a centrifugation temperature of 4℃. Take the upper plasma layer and freeze it at -80℃ for later use.

[0135] b. Plasma Sample Preparation: Add 1 μL of compound I or compound II to 100 μL of blank plasma and incubate with the stock solution at room temperature for 3 h to prepare a plasma sample with a mass concentration of 50 μg / mL. Take 100 μL of plasma sample, add 400 μL of methanol, vortex mix for 5 min, centrifuge at 12000×g for 10 min at 4℃, collect the upper organic layer, freeze-dry, and dissolve the residue in 100 μL of methanol. Centrifuge at 12000×g for 10 min at 4℃, and filter the supernatant through a 0.22 μm filter membrane for analysis.

[0136] c. On-machine testing

[0137] The treated plasma sample was placed in a sample bottle and analyzed using a liquid chromatography-mass spectrometry analyzer, Acquity UPLC H-Class coupled to a SQ Detector 2 mass spectrometer (Waters).

[0138] Chromatographic conditions: Column: ACQUITY UPLC BEH C18 (1.7 mm, 2.1 x 50 mm); flow rate: 0.50 mL / min; sample volume: 2.00 μL; column temperature: 30 °C; mobile phase A: water + 0.1% formic acid, mobile phase B: acetonitrile, gradient elution was used, elution program (see Table 1).

[0139] Table 1. ACQUITY UPLC H-Class automatic gradient elution program

[0140]

[0141] Mass spectrometry conditions: MS system: SQ Detector 2; ionization mode: ESI+ / ESI-; capillary voltage: 3000 V; ion source temperature: 150 °C; desolvation gas temperature: 500 °C; desolvation gas flow rate: 1000 L / Hr; gas curtain gas flow rate: 50 L / Hr; data acquisition and processing were performed using MassLynx V4.1 software.

[0142] LC-MS results showed that, at the cellular level, compound I was able to inhibit the proliferation of liver cancer cells. The proportion of compound I hydrolyzed to compound II after incubation with plasma was detected by LC-MS, and the peak area under the chromatogram curve was integrated, as shown in Figure 7 The conversion rate of the acetyl hydrolysis to hydroxyl was 88.66%, and the proportion without hydrolysis was 11.01%.

[0143] Table 2. Integration of compound chromatogram curve area

[0144]

[0145] The above merely describes specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be encompassed within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of a compound in the manufacture of a medicament for the treatment of liver cancer mediated by PGK1, wherein, The compound is selected from or a pharmaceutically acceptable salt thereof.

2. Use according to claim 1, characterized in that, The medicament includes a pharmaceutically acceptable carrier. The medicament includes a pharmaceutically acceptable carrier.

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